Method for reductive amination of a ketone using a mutated enzyme

ABSTRACT

Methods for chemically transforming compounds using a mutated enzyme are provided, and more particularly a method for the production of an amino acid from a target 2-ketoacid, the production of an amine from a target ketone and the production of an alcohol from a target ketone. The methods comprise creating a mutated enzyme that catalyzes the reductive amination or transamination of the target 2-ketoacid or ketone or the reduction of the ketone and providing the mutated enzyme in a reaction mixture comprising the target 2-ketoacid or ketone under conditions sufficient to permit the formation of the desired amino acid, amine or alcohol to thereby produce the amino acid, amine or alcohol.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.09/702,421, filed Oct. 31, 2000 (now abandoned), and claims the benefitof U.S. Provisional Application No. 60/288,378, filed May 3, 2001 (nowexpired), the entire disclosures of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

Enzymes are proteins that are capable of catalyzing chemicaltransformations. Enzymes position a substrate or substrates in anoptimal configuration and stabilize the transition state in the reactionpathway, thereby determining which of several potential chemicaltransformations actually occurs. Enzymes can be highly specific, both interms of the reaction that occurs and in their choice of substrate.Enzymes often accelerate reactions by factors of more than a million.Because of their specificity and catalytic power, enzymes areincreasingly being used for industrial applications.

One family of enzymes that is especially useful for industrialapplications is the family of oxidoreductase enzymes. Oxidoreductasescatalyze redox reactions, such as the reduction of aldehydes and ketonesto alcohols, the reductive amination of ketones, aldehydes, andketoacids to amines and amino acids, the reduction of disulfides tothiols, the reduction of alkenes to alkanes and the like. Thesereactions are normally reversible, and frequently the same enzymescatalyze the corresponding oxidation reactions. For example, alcoholdehydrogenases and carbonyl reductases catalyze both the reduction ofaldehydes and ketones to alcohols and the oxidation of alcohols toaldehydes and ketones. Amino acid dehydrogenases catalyze the oxidationof amino acids to 2-ketoacids and the reductive amination of 2-ketoacidsin the presence of ammonium salts to amino acids. Similarly, disulfidereductases catalyze the oxidation of thiols to disulfides or mixeddisulfides. Reduction and oxidation reactions are collectively referredto herein as “redox reactions.”

Some oxidoreductases can be used to produce fine and specialtychemicals, and are especially useful for producing chiral intermediatesin the pharmaceutical and agricultural industries. Oxidoreductases, likemany other enzymes, require other molecules, such as cofactors andcosubstrates, for optimal activity. For example, mixed function oxidasesuse nicotinamide cofactors as part of the complex catalysis of ahydroxylation reaction for the production of chiral alcohols.

Although a number of different enzymes are known, the development of newapplications for enzymes such as oxidoreductases requires an expandedsearch for new enzymes that catalyze specific reactions of interest. Forexample, amino acid dehydrogenases that reductively aminate certain2-ketoacids to naturally occurring L-amino acids are known, but nosuitable amino acid dehydrogenase has been identified for the productionof many non-naturally occurring amino acids. The enzyme catalyzedreductive amination of ketones that are not 2-ketoacids is comparativelyquite rare. Similarly, the stereoselective reduction of ketonescatalyzed by alcohol dehydrogenases, ketoreductases and carbonylreductases is known for certain ketones, but enzymes are not availablefor catalyzing this reaction with many desired target ketones.Transaminases are known that catalyze the transamination of many2-ketoacids to alpha-amino acids, but certain target 2-ketoacids,particularly those corresponding to non-naturally occurring amino acids,are transaminated poorly, if at all.

There are several known methods to generate potential enzymes thatcatalyze specific reactions of interest. For example, diversepopulations of enzymes can be found in microorganisms harvested fromdifferent environments. These microorganisms can be cultured, and theirDNA extracted, amplified by PCR, and cloned into a host for expressionof the enzymes. Alternatively, various molecular biology techniques,such as mutagenesis, shuffling, molecular breeding, and gene reassembly,can be used to create vast numbers of mutant versions of an enzymeencoded by a known gene. Examples of gene shuffling and molecularbreeding are described in U.S. Pat. No. 5,605,793; U.S. Pat. No.5,811,238; U.S. Pat. No. 5,830,721; U.S. Pat. No. 5,837,458; U.S. Pat.No. 5,965,408; U.S. Pat. No. 5,958,672; U.S. Pat. No. 6,001,574; andU.S. Pat. No. 6,117,679, all incorporated herein by reference. Examplesof methods for constructing large numbers of mutants are described inU.S. Pat. No. 6,001,574; U.S. Pat. No. 6,030,779; and U.S. Pat. No.6,054,267, also incorporated herein by reference.

Once potential enzymes that may be able to catalyze specific reactionsof interest have been generated, the enzymes are tested for activity onthe desired substrate, or target compound. Because many enzymes such asoxidoreductases require nicotinamide cofactors for optimal activity,detection of the oxidation or reduction of the cofactor can be used as asignal of enzyme activity.

Currently, the most common method of detecting enzymes usingnicotinamide cofactors involves the direct measurement of the cofactor.For example, as a carbonyl reductase reduces a carbonyl group, theconcomitant oxidation of reduced nicotinamide, i.e., the conversion of areduced form of nicotinamide adenine dinucleotide (NADH) to nicotinamideadenine dinucleotide (NAD⁺) or the conversion of a reduced form ofnicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamideadenine dinucleotide phosphate (NADP⁺) can be detected using theabsorbance of the reduced form of the cofactor. This reaction of thecofactor can be monitored in a solution by observing the decrease in theabsorbance of the solution at 340 nm using a spectrophotometer.Alternatively, during the carbonyl reductase-catalyzed oxidation of analcohol to the corresponding aldehyde or ketone, NAD⁺ is converted toNADH or NADP⁺ is converted to NADPH. The reduction of the cofactor canbe detected by monitoring the increase in absorbance at 340 nm,corresponding to the increase in concentration of reduced nicotinamidecofactor. Similarly, fluorescence measurements of nicotinamide cofactorscan be performed as well. Additionally, the change in concentration ofoxidized or reduced nicotinamide cofactor can be used to detect otherenzymes catalyzing cofactor-requiring reactions of interest.

Detection of enzymatic activity is often performed on many enzymesources for a particular reaction of interest in a process calledscreening. Often when screening, and particularly when carrying out highthroughput screening, mixtures of cells or cell lysates containingsuspended insoluble material are used as potential sources for newenzymes because clarification of the crude mixtures is operationallydifficult. The difficulty in using such crude mixtures for routinescreening for nicotinamide cofactor-using enzymes is that the reactionmixtures contain suspended solid material in the form of cells or celldebris. This insoluble material impedes the transmission of lightthrough the solution and causes high background readings in theabsorbance measurements of the cofactors. The crude mixtures alsocontain various cellular metabolites and biochemicals that absorb at 340nm, further compromising the accuracy of the measurements. These issuesare even more problematic when using high throughput screening methodsdue to the small volumes used in high density array formats such asmicrotiter plates or chips. Similarly, if fluorescence measurements arecarried out, detecting the emission of fluorescence is also impeded bythe presence of insoluble material.

As an alternative, the products of the desired enzymatic reaction can bedetected directly by chromatographic techniques. This method requiressampling each individual reaction followed by chromatographic separationof the reaction products, which may include alcohols, carbonylcompounds, and the like. Such a procedure is complex and time-consumingand is impractical for high throughput screening assays when many enzymesources are tested for the desired enzymatic activity.

SUMMARY OF THE INVENTION

The present invention provides novel methods for chemically transformingcompounds using a mutated enzyme. In one embodiment, the invention isdirected to a method for the production of an amino acid from a target2-ketoacid. The method comprises creating a mutated enzyme thatcatalyzes the reductive amination or transamination of the target2-ketoacid; and providing the mutated enzyme in a reaction mixturecomprising the target 2-ketoacid under conditions sufficient to permitthe formation of the amino acid to thereby produce the amino acid.

In another embodiment, the invention is directed to a method for theproduction of an amine from a target ketone. The method comprisescreating a mutated enzyme that catalyzes the reductive amination ortransamination of the target ketone; and providing the mutated enzyme ina reaction mixture comprising the target ketone under conditionssufficient to permit the formation of the amine to thereby produce theamine.

In yet another embodiment, the invention is directed to a method for theproduction of an alcohol from a target ketone. The method comprisescreating a mutated enzyme that catalyzes the reduction of the targetketone; and providing the mutated enzyme in a reaction mixturecomprising the target ketone under conditions sufficient to permit theformation of the alcohol to thereby produce the alcohol.

In the above embodiments, the mutated enzyme may be created by providingan existing enzyme and mutating the existing enzyme to produce themutated enzyme. The activity of the mutated enzyme on the target2-ketoacid or ketone is determined by contacting the mutated enzyme witha composition comprising the target 2-ketoacid or ketone and thereafterdetermining whether there is a change in the pH of the composition.Thereafter, it is determined whether the mutated enzyme has moreactivity than the existing enzyme on the target 2-ketoacid or ketone.The existing enzyme and/or mutated enzyme can be present in acomposition containing whole cells, cell extracts, cell lysates,mixtures containing insoluble cells, particulates, cellular debris, orthe like. Other compounds in the composition that also absorb light at340 nm do not interfere with the detection of enzymatic activity usingthe method of the present invention because the pH change can bedetermined, for example, by observing a color change at a differentwavelength. Further, the wavelength of the color change can be selectedby using an appropriate pH indicator.

DETAILED DESCRIPTION

The present invention is directed to methods for chemically transformingcompounds using a mutated enzyme. In particularly preferred embodiments,the invention is directed to a method for the production of an aminoacid from a target 2-ketoacid, the production of an amine from a targetketone and the production of an alcohol from a target ketone. Theinventive method comprises creating a mutated enzyme that catalyzes thereductive amination or transamination of the target 2-ketoacid or ketoneor the reduction of the target ketone and providing the mutated enzymein a reaction mixture comprising the target 2-ketoacid or ketone underconditions sufficient to permit the formation of the desired amino acid,amine or alcohol to thereby produce the amino acid, amine or alcohol.

As used herein, the terms “mutated” and “mutating” refer broadly to anyof a variety of molecular biology techniques, such as mutagenesis,shuffling, molecular breeding, and gene reassembly, that can be used tocreate vast numbers of mutant versions of an enzyme encoded by a knowngene. The activity of the mutated enzyme on the target compound isdetermined by contacting the mutated enzyme with a compositioncomprising the target compound and thereafter determining whether thereis a change in the pH of the composition. Thereafter it is determinedwhether the mutated enzyme has more activity than the existing enzyme onthe target compound.

By determining in which reactions the pH indicator undergoes a colorchange, enzymes with the desired enzymatic activity can be detectedeasily, even in a high throughput format, enabling the more facilediscovery of new enzymes, particularly oxidoreductases that catalyzeuseful redox reactions.

Non-limiting examples of sources of material that can be screened toobtain the existing enzyme include microorganisms, such as bacteria andyeast, which naturally express oxidoreductases, and genetically modifiedmicroorganisms, which express wild-type, modified or mutatedoxidoreductases. Examples of useful materials to be screened includecell lysates, mixtures of cells, cell extracts, environmental samplesand isolates, and the like. The material may be provided as a solution,a suspension, a dried mixture, a solid, or the like. As a solution orsuspension, the composition to be screened may be prepared and stored asa solution or as a suspension in liquid form. The composition may bemaintained at room temperature, at refrigerator temperatures, or frozen.As a solid or dried mixture, the composition may be prepared bylyophilization or evaporation of a liquid composition. Alternatively,the solid composition may be prepared by mixing solid ingredients suchas a cofactor, a pH indicator, and a target compound. When a solidcomposition is used in the practice of the present invention, the solidcomposition is normally redissolved or resuspended prior to use by theaddition of water or water containing buffer.

As used herein, “target compound” refers to a substance that is desiredto be acted upon by an enzyme as a substrate. Typical target compoundsinclude aldehydes, ketones, disulfides, thiols, ketoacids, amines, aminoacids, alcohols, alkenes, alkanes, and the like. In connection with theinventive methods, the term “target compound” does not include enzymesubstrates that undergo a hydrolytic transformation that results in thecreation or removal of an acidic or basic functionality, such as acarboxylic acid group. Target compounds are often chiral and/ortransformed into chiral compounds by enzymes, and enrichment in singlestereoisomers can occur.

As used herein, the term “pH indicator” means any material or substancethat changes its properties in response to a change in pH. Preferredchanges in properties include a change in optical properties, such as acolor change. Examples of pH indicators useful in the practice of thepresent invention include, but are not limited to, cresol red, m-cresolpurple, bromothymol blue, bromophenol red, bromophenol blue, phenol red,and phenolphthalein. The pH indicator can be selected independently foreach screen to determine the pH range or match a desired pH range forthe enzyme to be detected. For example, m-cresol purple is yellow at apH of about 7.4 and purple at a pH of about 9.0. Cresol red is yellow ata pH of about 7.2 and red at a pH of about 8.8. Bromothymol blue isyellow at a pH of about 6.0 and red at a pH of about 7.6. Bromophenolred is yellow at a pH of about 5.2 and red at a pH of about 6.8.Bromophenol blue is yellow at a pH of about 3.0 and blue at a pH ofabout 4.6. Phenol red is yellow at a pH of about 6.8 and red at a pH ofabout 8.2. Phenolphthalein is colorless at a pH of about 8.0 and pink ata pH of about 9.8. Other pH indicators can be selected depending on thedesired pH range for the reaction and the desired color change.

The conditions of the determination step can be adjusted to favor thedetection or screening of an enzyme with a desired pH optimum byadjusting the pH of the reaction mixture used in the screen. Forexample, when an amino acid dehydrogenase that functions at pH 6 issought, the reagent composition used for the screen can be buffered at apH of 6 using a buffer that has its optimum buffering capacity near pH6, and a pH indicator can be selected that changes color within therange of pH 5 to 7. Similarly, when an alcohol dehydrogenase thatcatalyzes the oxidation of a target alcohol at pH 9 is sought, thereagent composition used for the screen can be buffered at a pH of 9using a buffer that has its optimum buffering capacity near pH 9, and apH indicator can be selected that changes color within the range of pH 8to 10. Typically, the pH indicator is selected such that it exhibits acolor change in response to a change in pH within a range of about 1 to1.5 pH units on either side of the desired pH for the reaction.

If a buffer is used, the concentration of the buffer is preferablyadjusted in order to maintain a desired initial pH for the screeningreaction mixture and to reduce or eliminate small changes in pH notcaused by the desired redox reaction. However, the concentration of thebuffer should not be so high as to impede the change of pH that occursas the reaction catalyzed by the oxidoreductase proceeds. The buffer maybe any substance that helps maintain the desired initial pH of thesolution. Examples include potassium phosphate, sodium phosphate,potassium borate, sodium borate, sodium acetate, potassium acetate,sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassiumcarbonate, TRIS, PIPES, HEPES, MOPS, TEA, CHES, and the like. A listingof some useful biological buffers along with the pH ranges at which theyare most effective as buffers can be found in the Catalog ofBiochemicals and Reagents for Life Science Research; Sigma Chemical: St,Louis, 1998; p 1871. Often, desirable buffer concentrations must bedetermined experimentally. However, typical buffer concentrations usingthe method of the present invention are from about 0.01 mM to about 20mM, and preferably from about 0.05 mM to about 5 mM.

The above-described method can be used to determine activity of anyenzyme that causes a pH change when it catalyzes the reaction of atarget compound, and preferably is used to determine activity ofoxidoreductases. As used herein, “oxidoreductase” refers to an enzymecapable of performing an oxidation reaction or reduction reaction.Nonlimiting examples of oxidoreductases include a reductase, an oxidase,a dehydrogenase, a ketoreductase, an alcohol dehydrogenase, a carbonylreductase, an aldehyde dehydrogenase, an amino acid dehydrogenase, anamine oxidase, a disulfide reductase, an enoate reductase, and a mixedfunction oxidase. A listing of such enzymes can be found in EnzymeNomenclature; Webb, E. C., Ed. Academic: Orlando 1984; pp 20–141, thedisclosure of which is incorporated herein by reference.

Often enzymes, and particularly oxidoreductases, require cofactors orcosubstrates for optimal activity. As used herein, the term “cofactor”means any molecule that participates in a chemical transformation of thetarget compound, including cofactors and cosubstrates. Nonlimitingexamples of cofactors include nicotinamide cofactors, flavins, andderivatives and analogs thereof.

As used herein, “nicotinamide cofactor” refers to any type of theoxidized and reduced forms of nicotinamide adenine dinucleotide (NAD⁺and NADH, respectively) and the oxidized and reduced forms ofnicotinamide adenine dinucleotide phosphate (NADP⁺ and NADPH,respectively) and derivatives and analogs thereof. With regard to anicotinamide cofactor, the term “derivative” means any compoundcontaining a pyridine structural element, including nicotinamides thathave been chemically modified by attachment to soluble or insolublepolymeric materials. Some examples of derivatives of nicotinamidecofactors are described in U.S. Pat. No. 5,106,740, and Mansson andMosbach Methods in Enzymology (1987) 136, 3–45, the disclosures of whichare incorporated herein by reference. The term “analogs,” as usedherein, refers to materials that undergo a formal hydride transfer in aredox reaction similar to that undergone by nicotinamide cofactors.Examples of analogs of nicotinamide cofactors useful in the practice ofthe present invention include compounds described in U.S. Pat. No.5,801,006, the disclosure of which is incorporated herein by reference.Other suitable cofactors, as defined herein, can be used in the practiceof the invention, as would be recognized by those skilled in the art.

In the practice of the invention, the nicotinamide cofactors can be usedin equimolar quantities relative to the target ketone, alcohol, amine oramino acid, or the cofactors may be recycled, if desired. Numerousmethods for the recycling of nicotinamide cofactors are well-known inthe art, and any of these methods may be used in the practice of thepresent invention. Some of the methods for recycling nicotinamidecofactors are described in G. L. Lemiere, et al., Tetrahedron Letters,26, 4257 (1985); in “Enzymes as Catalysts for Organic Synthesis,” pp.19–34, M. Schneider, Ed., Reidel Dordecht, 1986; in Z. Shaked and G. M.Whitesides, J. Am. Chem. Soc. 102, 7104–5 (1980); and J. B. Jones and T.Takamura, Can. J. Chem. 62, 77 (1984); the disclosures of which areincorporated herein by reference. In the use of these recycling methods,an amount of about 0.0001 mole to about 0.05 mole of nicotinamidecofactor is used per mole of ketone to be reduced or reductivelyaminated, per mole of 2-ketoacid to be reductively aminated, or per moleof alcohol or amine or amino acid to be oxidized, providing a recyclenumber for the cofactor of from about 20 to about 10,000.

As an example of an oxidoreductase-catalyzed transformation, thereaction for the reduction of a ketone to a chiral alcohol is shown inScheme 1.

In the above scheme, and the schemes set forth below, A and B areindependently selected from hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, heterocyclic, and the like. As shown in the balancedequation in Scheme 1, one proton is consumed in the reaction for eachmolecule of nicotinamide cofactor oxidized and each molecule of alcoholformed. As the reaction progresses, the consumption of protons causesthe pH of the reaction mixture to rise. By including a suitable pHindicator in the reaction mixture, the presence of an alcoholdehydrogenase is indicated by a change in color of the reaction mixture.Although positive reactions can be detected spectrophotometrically, ifdesired, the use of a colorimetric pH indicator has the added advantagethat the presence of oxidoreductase enzymes can be detected visually andwithout expensive instrumentation.

Because of the reversibility of most reactions catalyzed byoxidoreductases, an oxidation reaction can also be used for screeningand detection. For example, an alcohol dehydrogenase or carbonylreductase catalyzes the oxidation of an alcohol to form an oxidizedcarbonyl compound, shown in Scheme 2.

Thus, the presence of an oxidoreductase catalyzing this reaction can bedetected using a reaction mixture containing an oxidized nicotinamidecofactor, an alcohol, and a pH indicator. In this case, the pH of thereaction mixture will decrease as the reaction progresses, and thedecrease in pH is detected by the change in color of the pH indicator.

As used herein, the term “carbonyl compound” means any chemical compoundthat has incorporated into it a functional group consisting of acarbon-oxygen double bond. The terms “carbonyl reductase”,“ketoreductase”, and “alcohol dehydrogenase” mean any enzyme that cancatalyze the chemical reduction of a carbonyl group in the presence of anicotinamide cofactor.

With oxidoreductases that produce amines and amino acids by reductiveamination, ammonia or ammonium ion is also a reactant. Thus, whenscreening for an amine or amino acid dehydrogenase using the method ofthe present invention, ammonia or a salt of ammonium ion is alsoincluded with a pH indicator, a reduced nicotinamide cofactor, and aketone or ketoacid to be reductively aminated. The reaction is shown inScheme 3.

For detection of an amine or amino acid dehydrogenase that can oxidizean amine or amino acid to the corresponding ketone or ketoacid usingthen method of the present invention, the reaction mixture for detectioncontains a pH indicator, an oxidized nicotinamide cofactor, and an amineor amino acid to be oxidized. The reaction for the oxidation of an amineor amino acid using an amine or amino acid dehydrogenase is depicted inScheme 4:

The above-described method can be used to detect enzymes using a secondreaction that can be coupled to the enzyme-catalyzed reaction to bescreened. For example, in screening for an aminotransferase thatcatalyzes the transamination of a ketone or ketoacid with L-asparticacid as the donor, the reaction products are an amine or amino acid andoxaloacetate. In a second reaction, the oxaloacetate can bedecarboxylated to pyruvate, with the consumption of a proton. Thus,aminotransferase activity can be detected by detecting an increase inthe pH of the reaction mixture because the decarboxylation ofoxaloacetate is coupled to the transamination reaction to be screened.

This method is particularly useful for screening for enzymes to performspecific chemical transformations of target compounds that areintermediates in chemical syntheses. Thus, after an enzyme has beendetermined to have activity for a particular target compound, it can beused to convert that target compound to a useful chemical intermediate,as described above. Useful chemical intermediates include alcohols,amines, alpha-amino acids, beta-amino acids, gamma-amino acids,aldehydes, ketones, carboxylic acids, esters, amides, and the like.

As discussed above, enzymes often require cofactors or cosubstrates foroptimal activity. Accordingly, when converting a target compound,preferably a cofactor is present with the enzyme. Suitable cofactors areset forth above.

In the pharmaceutical industry, it is often desirable to chemicallytransform target compounds into one stereoisomer to the substantialexclusion of another. More specifically, it is desirable to obtain thesecompounds in more than about 90% enantiomeric excess (ee), preferably inabout 95% ee, and still more preferably in about 98% ee, because of theconsiderable difficulty and the tremendous waste of material inseparating enantiomeric products from a racemic mixture. Because enzymescan perform chemical transformations exclusively forming oneenantiomeric product and often are easier to use and more cost-effectivethan performing an asymmetric synthesis, new enzymes that can act upontarget compounds are sought after, such as a carbonyl reductase thatproduces of a single stereoisomer of a alcohol in 98% ee.

In a particularly preferred embodiment, the target compound is a ketonethat is not a ketoacid, and the target compound is converted to anamine, preferably a chiral amine, in the presence of an aminedehydrogenase. Preferably the above-described screening method is usedto first determine enzymatically-active amino acid dehydrogenases.Further screening is then performed on the enzymatically-active aminoacid dehydrogenases, again, in accordance with the procedures describedabove, to identify enzymatically-active amine dehydrogenases. The thusidentified enzymatically-active amine dehydrogenase is then provided ina solution together with a ketone, ammonia and reduced nicotinamidecofactor to synthesize an amine.

The invention is now further described by the following examples, whichare given here for illustrative purposes only and are not intended tolimit the scope of the invention.

EXAMPLES Example 1 General Procedure for Detection of an Enzyme thatReduces the Target Compound ethyl-4-chloro-3-ketobutyrate

A gene encoding the alcohol dehydrogenase YPR1 (described by Nakamura,K., et al., Bioscience, Biotechnology and Biochemistry, (1997) 61,375–377), is subjected to mutagenesis by error-prone PCR according tothe method of May, O., et al., (Nature Biotechnology, (2000) 18,317–320). The error-prone PCR is performed in a 100 mL reaction mixturecontaining 0.25 ng of plasmid DNA as a template dissolved in PCR buffer(10 mM TRIS, 1.5 mM MgCl₂, 50 mM KCl, pH 8.3), and also containing 0.2mM of each dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase(Roche Diagnostics, Indianapolis, Ind.). Conditions for carrying out thePCR are as follows: 2 minutes at 94° C.; 30 cycles of 30 sec 94° C., 30sec 55° C.; 2 minutes at 72° C. The PCR product is double digested withNco I and Bgl II and subcloned into pBAD/HisA vector (Invitrogen,Carlsbad, Calif.) which has been digested with the same restrictionenzymes. The resulting YPR1 mutant library is transformed into the E.coli host strain LMG194 (Invitrogen, Carlsbad, Calif.) and plated on LBagar supplied with 100 μg/mL ampicillin. Individual transformants areinoculated into 96-well microtiter plates (hereafter referred to asmaster plates) containing 0.2 mL LB Broth with 100 μg/mL ampicillin, andgrowth is allowed to take place for 8 to 16 hours at 37° C. with shakingat 200 rpm. Each well in each master plate is then re-inoculated by areplica plating technique into a new second stage 96-well platepre-loaded with the same growth media plus 2 g/L of arabinose, andgrowth is allowed to continue for 5–10 hours at 37° C. with shaking at200 rpm. The second stage plates are then centrifuged at 14,000 rpm for20 minutes, and the supernatant is decanted. The cell pellet in eachwell is washed with 200 mL of water. The washed cell pellet is suspendedin 30 mL of B-Per Bacterial Protein Extraction Reagent (Pierce ChemicalCo., Rockford, Ill.), hereinafter “B-Per.” After mixing, the suspensionof cells in B-Per reagent is allowed to stand for 10 minutes at roomtemperature, and a reaction solution having the following composition isthen added to each well in the plate:

-   -   7.5 μL of a pH 6.5 solution containing 8 μg/mL of NADPH    -   7.5 μL of a pH 6.5 50% DMSO solution containing 0.25 M        ethyl-4-chloro-3-ketobutyrate    -   155 μL of 1 mM potassium phosphate buffer, pH 6.5    -   1.5 μL of a 4 μg/mL solution of cresol red indicator        Wells containing an alcohol dehydrogenase that catalyzes the        reduction of the target compound ethyl-4-chloro-3-ketobutyrate        can be identified easily as their color changes from an initial        yellow to an orange or red color. These wells are correlated to        the original wells in the master plates to obtain the original        clones of mutant alcohol dehydrogenase that catalyzes the        desired reduction reaction.

Example 2 Detection of an Enzyme that Reduces the Target Compoundethyl-3-phenyl-3-ketopropionate

The procedure of Example 1 is repeated, replacing theethyl-4-chloro-3-ketobutyrate with ethyl-3-phenyl-3-ketopropionate,thereby identifying at least one mutant alcohol dehydrogenase thatcatalyzes the desired reduction reaction.

Example 3 Detection of an Enzyme that Reduces the Target Compoundethyl-indan-2-one-1-carboxylate

The procedure of Example 1 is repeated, replacing theethyl-4-chloro-3-ketobutyrate with ethyl-indan-2-one-1-carboxylate,thereby identifying at least one mutant alcohol dehydrogenase thatcatalyzes the desired reduction reaction.

Example 4 Detection of an Enzyme that Reduces the Target Compoundethyl-4-phenyl-4-ketobutyrate

The procedure of Example 1 is repeated, replacing theethyl-4-chloro-3-ketobutyrate with ethyl-4-phenyl-4-ketobutyrate,thereby identifying at least one mutant alcohol dehydrogenase thatcatalyzes the desired reduction reaction.

Example 5 Detection of an Enzyme that Reduces the Target Compoundethyl-3-phenyl-3-ketopropionate

The procedure of Example 1 is repeated, replacing the reaction solutionwith a reaction solution of the following composition:

-   -   7.5 μL of a pH 6.5 solution containing 8 μg/mL of NADPH    -   7.5 μL of pH 6.5 DMSO solution containing 0.25 M        ethyl-3-phenyl-3-ketopropionate    -   155 μL of a 1 mM potassium phosphate buffer, pH 7.0    -   1.5 μL of a 4 μg/mL solution of a cresol red indicator        Wells containing an alcohol dehydrogenase that reduces        ethyl-3-phenyl-3-ketopropionate can be identified easily as the        color changes from an initial yellow to a red color. At least        one mutant alcohol dehydrogenase that catalyzes the desired        reduction reaction is identified.

Example 6 Detection of an Enzyme that Reduces the Target Compoundethyl-indan-2-one-1-carboxylate

The procedure of Example 5 is repeated, replacing theethyl-3-phenyl-3-ketopropionate with ethyl-indan-2-one-1-carboxylate,thereby identifying at least one mutant alcohol dehydrogenase thatcatalyzes the desired reduction reaction.

Example 7 Detection of an Enzyme that Reduces the Target Compoundethyl-4-phenyl-4-ketobutyrate

The procedure of Example 5 is repeated, replacing theethyl-3-phenyl-3-ketopropionate with ethyl-4-phenyl-4-ketobutyrate,thereby identifying at least one mutant alcohol dehydrogenase thatcatalyzes the desired reduction reaction.

Example 8 Detection of an Enzyme that Reduces the Target Compoundethyl-cyclohexanone-2-carboxylate

The procedure of Example 5 is repeated, replacing theethyl-3-phenyl-3-ketoproponiate with ethyl-cyclohexanone-2-carboxylate,thereby identifying at least one mutant alcohol dehydrogenase thatcatalyzes the desired reduction reaction.

Example 9 Detection of an Enzyme that Reduces the Target Compoundethyl-2-ethyl-3-ketobutyrate

The procedure of Example 1 is repeated, replacing the reaction solutionwith a reaction solution of the following composition:

-   -   7.5 μL of a pH 6.5 solution containing 8 μg/mL of NADPH    -   7.5 μL of a pH 6.5 50% DMSO solution containing 0.25 M        ethyl-2-ethyl-3-ketobutyrate    -   155 μL of a 2 mM potassium phosphate buffer, pH 6.5    -   1.5 μL of a 4 μg/mL solution of bromothymol blue indicator        Wells containing an alcohol dehydrogenase that reduces        ethyl-2-ethyl-3-ketobutyrate can be identified easily as the        color changes from an initial yellow to a blue color.

Example 10 Detection of an Enzyme that Reduces the Target Compoundethyl-2-allyl-3-ketobutyrate

The procedure of Example 9 is repeated, replacing theethyl-2-ethyl-3-ketobutyrate with ethyl-2-allyl-3-ketobutyrate, therebyidentifying at least one mutant alcohol dehydrogenase that catalyzes thedesired reduction reaction.

Example 11 Detection of an Enzyme that Reduces the Target Compoundethyl-2-phenyl-3-ketobutyrate

The procedure of Example 9 is repeated, replacing theethyl-2-ethyl-3-ketobutyrate with ethyl-2-phenyl-3-ketobutyrate, therebyidentifying at least one mutant alcohol dehydrogenase that catalyzes thedesired reduction reaction.

Example 12 Detection of an Enzyme that Reduces the Target Compoundethyl-2-benzyl-3-ketobutyrate

The procedure of Example 9 is repeated, replacing theethyl-2-ethyl-3-ketobutyrate with ethyl-2-benzyl-3-ketobutyrate, therebyidentifying at least one mutant alcohol dehydrogenase that catalyzes thedesired reduction reaction.

Example 13 Detection of an Enzyme that Oxidizes a Target CompoundAlcohol

A gene encoding the alcohol dehydrogenase Alr1 (Yamada, et al., FEMSMicrobiology Letters, (1990) 70, 45–48) is subjected to mutagenesis byerror-prone PCR according to the method of May et al. The error-pronePCR is performed in a 100 mL reaction mixture containing 0.25 ng ofplasmid DNA as template dissolved in PCR buffer containing 0.2 mM ofeach dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase.Conditions for carrying out the PCR are as follows: 2 minutes at 94° C.;30 cycles of 30 sec 94° C., 30 sec 55° C.; 2 minutes at 72° C. The PCRproduct is double digested with Nco I and Bgl II and subcloned intopBAD/HisA vector which has been digested with the same restrictionenzymes. The resulting Alr1 mutant library is transformed into an E.coli host strain LMG194 and plated on LB agar supplied with 100 μg/mLampicillin. Individual transformants are inoculated into master platescontaining 0.2 mL LB Broth with 100 μg/mL ampicillin, and growth isallowed to take place for 8–16 hours at 37° C. with shaking at 200 rpm.Each well in each master plate is then re-inoculated by a replicaplating technique into a new second stage 96-well plate pre-loaded withthe same growth media plus 2 g/L of arabinose, and growth is allowed tocontinue for 5 to 10 hours at 37° C. with shaking at 200 rpm. The secondstage plates are then centrifuged at 14,000 rpm for 20 minutes, and thesupernatant is decanted. The cell pellet in each well is washed with 200mL of water. The washed cell pellet is suspended in 30 mL of B-Per.After mixing, the suspension of cells in B-Per reagent is allowed tostand for 10 minutes at room temperature, and a solution having thefollowing composition is then added to each well in the plate:

-   -   7.5 μL of a pH 8.0 solution containing 20 μg/ml of NADP⁺    -   7.5 μL of a pH 8.0 50% DMSO solution containing 0.25 M        (2R,3S)-ethyl-2-ethyl-3-hydroxybutyrate    -   155 μL of 2 mM potassium phosphate buffer, pH 8.0    -   1.5 μL of a 4 μg/ml solution of bromothymol blue indicator        Wells in which the color changes from an initial blue to a        yellow color contain mutant alcohol dehydrogenases that catalyze        the oxidation of the target alcohol        (2R,3S)-ethyl-2-ethyl-3-hydroxybutyrate. These wells are        correlated to the original wells in the master plates to obtain        the original clones of mutant alcohol dehydrogenases catalyzing        the desired oxidation reaction.

Example 14 General Procedure for Detection of an Enzyme that ReductivelyAminates the Target Compound 3,3-dimethyl-2-ketobutyrate

A gene encoding leucine dehydrogenase from B. stearothermophilus(Nagata, et al. Biochemistry (1998) 27, 9056) is subjected tomutagenesis by error-prone PCR according to the method of May et al. Theerror-prone PCR is performed in a 100 mL reaction mixture containing0.25 ng of plasmid DNA as template dissolved in PCR buffer alsocontaining 0.2 mM of each dNTP, 50 pmol of each primer and 2.5 units ofTaq polymerase. Conditions for carrying out the PCR are as follows: 2minutes at 94° C.; 30 cycles of 30 sec 94° C., 30 sec 55° C.; 2 minutesat 72° C. The PCR product is double digested with Nco I and Bgl II andsubcloned into pBAD/HisA vector which has been digested with the samerestriction enzymes. The resulting leucine dehydrogenase mutant libraryis transformed into an E. coli host strain LMG194 and plated on LB agarsupplied with 100 μg/mL ampicillin. Individual transformants areinoculated into master plates containing 0.2 mL LB Broth with 100 μg/mLampicillin, and growth is allowed to take place for 8 to 16 hours at 37°C. with shaking at 200 rpm. Each well in each master plate is thenre-inoculated by a replica plating technique into a new second stage96-well plate pre-loaded with the same growth media plus 2 g/L ofarabinose, and growth is allowed to continue for 5–10 hours at 37° C.with shaking at 200 rpm. The second stage plates are then centrifuged at14,000 rpm for 20 minutes, and the supernatant is decanted. The cellpellet in each well is washed with 200 mL of water. The washed cellpellet is suspended in 30 mL of B-Per After mixing, the suspension ofcells in B-Per reagent is allowed to stand for 10 minutes at roomtemperature, and a solution having the following composition is thenadded to each well in the plate:

-   -   7.5 μL of a pH 6.5 solution containing 8 μg/mL of NADH    -   7.5 μL of a pH 6.5 50% DMSO solution containing 0.25 M        3,3-dimethyl-2-ketobutyrate    -   155 μL f 1 mM potassium phosphate buffer, pH 6.5, containing 100        mM ammonium chloride    -   1.5 μL of a 4 μg/mL solution of cresol red indicator        Wells in which the color changes from an initial yellow to an        orange or red color contain leucine dehydrogenase that catalyzes        the reductive amination of the target 2-ketoacid        3,3-dimethyl-2-ketobutyrate. These wells are correlated to the        original wells in the master plates to obtain the original        clones of mutant leucine dehydrogenase that catalyzes the        desired reductive amination reaction.

Example 15 Detection of an Enzyme that Reductively Aminates the TargetCompound 4-(methylphosphinyl)-2-ketobutyrate

The procedure of Example 14 is repeated, replacing3,3-dimethyl-2-ketobutyrate with 4-(methylphosphinyl)-2-ketobutyrat,thereby identifying at least one mutant dehydrogenase that catalyzes thedesired reductive amination reaction.

Example 16 Detection of an Enzyme that Reductively Aminates the TargetCompound 3-(2-naphthyl)pyruvate

The procedure of Example 14 is repeated, replacing3,3-dimethyl-2-ketobutyrate with 3-(2-naphthyl)pyruvate, therebyidentifying at least one mutant dehydrogenase that catalyzes the desiredreductive amination reaction.

Example 17 Detection of an Enzyme that Reductively Aminates the TargetCompounds 3-(1-naphthyl)pyruvate

The procedure of Example 14 is repeated, replacing the3,3-dimethyl-2-ketobutyrate with 3-(1-naphthyl)pyruvate, therebyidentifying at least one mutant dehydrogenase that catalyzes the desiredreductive amination reaction.

Example 18 Detection of an Enzyme that Reductively Aminates the TargetCompound 4-phenyl-2-ketobutyrate

The procedure of Example 14 is repeated, replacing3,3-dimethyl-2-ketobutyrate with 4-phenyl-2-ketobutyrate, therebyidentifying at least one mutant dehydrogenase that catalyzes the desiredreductive amination reaction.

Example 19 Detection of an Enzyme that Reductively Aminates the TargetCompound 4,4-dimethyl-2-ketopentanoate

The procedure of Example 14 is repeated replacing the3,3-dimethyl-2-ketobutyrate with 4,4-dimethyl-2-ketopentanoate, therebyidentifying at least one mutant dehydrogenase that catalyzes the desiredreduction reaction.

Example 20 Detection of an Enzyme that Oxidizes the Target CompoundL-tert-leucine

A gene encoding the leucine dehydrogenase from B. stearothermophilus issubjected to mutagenesis by error-prone PCR according to the method ofMay et al. The error-prone PCR is performed in a 100 mL reaction mixturecontaining 0.25 ng of plasmid DNA as template dissolved in PCR buffer(10 mM TRIS, 1.5 mM MgCl₂, 50 mM KCl, pH 8.3), and also containing 0.2mM of each dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase.Conditions for carrying out the PCR are as follows: 2 minutes at 94° C.;30 cycles of 30 sec 94° C., 30 sec 55° C.; 2 minutes at 72° C. The PCRproduct is double digested with Nco I and Bgl II and subcloned intopBAD/HisA vector which has been digested with the same restrictionenzymes. The resulting leucine dehydrogenase mutant library istransformed into an E. coli host strain LMG194 and plated on LB agarsupplied with 100 μg/mL ampicillin. Individual transformants areinoculated into 96-well master plates containing 0.2 mL LB Broth with100 μg/mL ampicillin, and growth is allowed to take place for 8–16 hoursat 37° C. with shaking at 200 rpm. Each well in each master plate isthen re-inoculated by a replica plating technique into a new secondstage 96-well plate pre-loaded with the same growth media plus 2 g/L ofarabinose, and growth is allowed to continue for 5–10 hours at 37° C.with shaking at 200 rpm. The second stage plates are then centrifuged at14,000 rpm for 20 minutes, and the supernatant is decanted. The cellpellet in each well is washed with 200 mL of water. The washed cellpellet is suspended in 30 mL of B-Per. After mixing, the suspension ofcells in B-Per reagent is allowed to stand for 10 minutes at roomtemperature, and a solution having the following composition is thenadded to each well in the plate:

-   -   7.5 μL of a pH 8.0 solution containing 20 μg/ml of NAD⁺    -   7.5 μL of a pH 8.0 50% DMSO solution containing 0.25 M        L-tert-leucine (S-3,3-dimethyl-2-aminobutyrate)    -   155 μL of 2 mM potassium phosphate buffer, pH 8.0, containing        100 mM ammonium chloride    -   1.5 μL of a 4 μg/ml solution of bromothymol blue indicator        Wells in which the color changes from an initial blue to a        yellow color contain mutant leucine dehydrogenases that catalyze        the oxidation of the target amino acid. These wells are        correlated to the original wells in the master plates to obtain        the original clones of mutant leucine dehydrogenases catalyzing        the desired oxidation reaction.

Example 21 Detection of an Enzyme that Oxidizes the Target CompoundS-phosphinothricin

The procedure of Example 20 is repeated replacing the L-tert-leucinewith S-phosphinothricin, thereby identifying at least one mutant alcoholdehydrogenase that catalyzes the desired oxidation reaction.

Example 22 Detection of an Enzyme that Oxidizes the Target CompoundS-(2-naphthyl)alanine

The procedure of Example 20 is repeated, replacing the L-tert-leucinewith S-(2-naphthyl)alanine, thereby identifying at least one mutantalcohol dehydrogenase that catalyzes the desired oxidation reaction.

Example 23 Detection of an Enzyme that Oxidizes the Target CompoundD-tert-leucine

The procedure of Example 20 is repeated, replacing the L-tert-leucinewith D-tert-leucine, thereby identifying at least one mutant alcoholdehydrogenase that catalyzes the desired oxidation reaction.

Example 24 Detection of an Enzyme that Oxidizes the Target CompoundS-4-phenyl-2-aminobutyrate

The procedure of Example 20 is repeated, replacing L-tert-leucine withS-4-phenyl-2-aminobutyrate, thereby identifying at least one mutantalcohol dehydrogenase that catalyzes the desired oxidation reaction.

Example 25 Detection of an Enzyme that Oxidizes the Target CompoundD-tyrosine

The procedure of Example 20 is repeated, replacing the L-tert-leucinewith D-tyrosine, thereby identifying at lest one mutant alcoholdehydrogenase that catalyzes the desired oxidation reaction.

Example 26 General Procedure for Detection of an Enzyme Mutant thatReductively Aminates the Target Compound Acetophenone

A gene encoding leucine dehydrogenase from B. stearothermophilus issubjected to mutagenesis by error-prone PCR according to the method ofMay, et al. The error-prone PCR is performed in a 100 mL reactionmixture containing 0.25 ng of plasmid DNA as template dissolved in PCRbuffer containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5units of Taq polymerase. Conditions for carrying out the PCR are asfollows: 2 minutes at 94° C.; 30 cycles of 30 sec 94° C., 30 sec 55° C.;2 minutes at 72° C. The PCR product is double digest with Nco I and BglII and subcloned into pBAD/HisA vector which has been digested with thesame restriction enzymes. The resulting leucine dehydrogenase mutantlibrary is transformed into an E. coli host strain LMG194 and plated onLB agar supplied with 100 μg/mL ampicillin. Individual transformants areinoculated into master plates containing 0.2 mL LB Broth with 100 μg/mLampicillin, and growth is allowed to take place for 8–16 hours at 37° C.with shaking at 200 rpm. Each well in each master plate is thenre-inoculated by a replica plating technique into a new second stage96-well plate pre-loaded with the same growth media plus 2 g/L ofarabinose, and growth is allowed to continue for 5–10 hours at 37° C.with shaking at 200 rpm. The second stage plates are then centrifuged at14,000 rpm for 20 minutes, and the supernatant is decanted. The cellpellet in each well is washed with 200 mL of water. The washed cellpellet is suspended in 30 mL of B-Per. After mixing, the suspension ofcells in B-Per reagent is allowed to stand for 10 minutes at roomtemperature, and a solution having the following composition is thenadded to each well in the plate:

-   -   7.5 μL of a pH 6.5 solution containing 8 μg/mL of NADH    -   7.5 μL of a pH 6.5 50% DMSO solution containing 0.25 M        acetophenone    -   155 μL of 1 mM potassium phosphate buffer, pH 6.5, containing        100 mM ammonium chloride    -   1.5 μL of a 4 μg/mL solution of cresol red indicator        Wells in which the color change from an initial yellow to an        orange or red color contain leucine dehydrogenases that        catalyzes the reductive amination of the target ketone        acetophenone. These wells are correlated to the original wells        in the master plates to obtain the original clones of mutant        leucine dehydrogenase that catalyzes the desired reductive        amination reaction.

Example 27 Detection of an Enzyme that Oxidizes the Target CompoundS-1-phenylethylamine

A gene encoding the leucine dehydrogenase from B. stearothermophilus issubjected to mutagenesis by error-prone PCR according to the method ofMay, et al. The error-prone PCR is performed in a 100 mL reactionmixture containing 0.25 ng of plasmid DNA as template dissolved in PCRbuffer containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5units of Taq polymerase. Conditions for carrying out the PCR are asfollows: 2 minutes at 94° C.; 30 cycles of 30 sec 94° C., 30 sec 55° C.;2 minutes at 72° C. The PCR product is double digest with Nco I and BglII and subcloned into pBAD/HisA vector which has been digested with thesame restriction enzymes. The resulting leucine dehydrogenase mutantlibrary is transformed into an E. coli host strain LMG194 and plated onLB agar supplied with 100 μg/mL ampicillin. Individual transformants areinoculated into master plates containing 0.2 mL LB Broth with 100 μg/mLampicillin, and growth is allowed to take place for 8–16 hours at 37° C.with shaking at 200 rpm. Each well in each master plate is thenre-inoculated by a replica plating technique into a new second stageplate pre-loaded with the same growth media plus 2 g/L of arabinose, andgrowth is allowed to continue for 5 to 10 hours at 37° C. with shakingat 200 rpm. The second stage plates are then centrifuged at 14,000 rpmfor 20 minutes, and the supernatant is decanted. The cell pellet in eachwell is washed with 200 mL of water. The washed cell pellet is suspendedin 30 mL of B-Per Bacterial Protein Extraction Reagent. After mixing,the suspension of cells in B-Per reagent is allowed to stand for 10minutes at room temperature, and a solution having the followingcomposition is then added to each well in the plate:

-   -   7.5 μL of a pH 8.0 solution containing 20 μg/mL of NAD⁺    -   7.5 μL of a pH 8.0 50% DMSO solution containing 0.25 M        S-1-phenylethylamine    -   155 μL of 2 mM potassium phosphate buffer, pH 8.0, containing        100 mM ammonium chloride    -   1.5 μL of a 4 μg/mL solution of bromothymol blue indicator        Wells in which the color changes from blue initially to yellow        contain mutant leucine dehydrogenase that catalyze the oxidation        of the target amine. These wells are correlated to the original        wells in the master plates to obtain the original clones of        mutant leucine dehydrogenases catalyzing the desired oxidation        reaction.

Example 28 Detection of an Enzyme that Oxidizes the Target CompoundR-1-phenylethylamine

A gene encoding the leucine dehydrogenase from B. stearothermophilus issubjected to mutagenesis by error-prone PCR according to the method ofMay, et al. The error-prone PCR is performed in a 100 mL reactionmixture containing 0.25 ng of plasmid DNA as template dissolved in PCRbuffer containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5units of Taq polymerase. Conditions for carrying out the PCR are asfollows: 2 minutes at 94° C.; 30 cycles of 30 sec 94° C., 30 sec 55° C.;2 minutes at 72° C. The PCR product is double digest with Nco I and BglII and subcloned into pBAD/HisA vector which has been digested with thesame restriction enzymes. The resulting leucine dehydrogenase mutantlibrary is transformed into an E. coli host strain LMG194 and plated onLB agar supplied with 100 μg/mL ampicillin. Individual transformants areinoculated into master plates containing 0.2 mL LB Broth with 100 μg/mLampicillin, and growth is allowed to take place for 8–16 hours at 37° C.with shaking at 200 rpm. Each well in each master plate is thenre-inoculated by a replica plating technique into a new second stageplate pre-loaded with the same growth media plus 2 g/L of arabinose, andgrowth is allowed to continue for 5–10 hours at 37° C. with shaking at200 rpm. The second stage plates are then centrifuged at 14,000 rpm for20 minutes, and the supernatant is decanted. The cell pellet in eachwell is washed with 200 mL of water. The washed cell pellet is suspendedin 30 mL of B-Per. After mixing, the suspension of cells in B-Perreagent is allowed to stand for 10 minutes at room temperature, and asolution having the following composition is then added to each well inthe plate:

-   -   7.5 μL of a pH 8.0 solution containing 20 μg/mL of NAD⁺    -   7.5 μL of a pH 8.0 50% DMSO solution containing 0.25 M        R-1-phenylethylamine    -   155 μL of 2 mM potassium phosphate buffer, pH 8.0, containing        100 mM ammonium chloride    -   1.5 μL of a 4 μg/mL solution of bromothymol blue indicator        Wells in which the color changes from blue initially to yellow        contain mutant leucine dehydrogenase that catalyze the oxidation        of the target amine. These wells are correlated to the original        wells in the master plates to obtain the original clones of        mutant leucine dehydrogenases catalyzing the desired oxidation        reaction.

Example 29 Production of 1-phenylethylamine by the Reductive Aminationof Acetophenone

One hundred units of an amine dehydrogenase generated by mutagenesis andscreening of leucine dehydrogenase as described in any one of Examples26 to 28 above is incubated at 45° C. in 100 milliliters of a solutionmaintained at pH 6.5 containing potassium phosphate (1 millimole), NADH(0.01 millimole), ammonium formate (25 millimoles), and formatedehydrogenase from Candida boidinii (100 units). Acetophenone (10millimoles) is added slowly over one hour with stirring, and thereaction is allowed to proceed for an additional 4 hours. Afterbasification of the reaction mixture to pH 12 and extraction with methylt-butyl ether, analysis of the reaction products is carried out by gaschromatography to determine the yield of 1-phenylethylamine. Chiralanalysis is carried out by chiral gas chrmoatography using a ChiraDex CBcolumn (Advanced Separation Technology, Whippany, N.J. USA).

Example 30 Production of R-1-phenylethylamine by the Reductive Aminationof Acetophenone

The method of Example 29 is carried out except that the aminedehydrogenase is an R-1-phenylethylamine dehydrogenase and the productis R-1-phenylethylamine.

Example 31 Production of S-1-phenylethylamine by the Reductive Aminationof Acetophenone

The method of Example 29 is carried out except that the aminedehydrogenase is an S-1-phenylethylamine dehydrogenase and the productis S-1-phenylethylamine.

Example 32 Production of R-1-(p-chlorophenyl)ethylamine by the ReductiveAmination of p-chloroacetophenone

The method of Example 30 is carried out except that acetophenone isreplaced by p-chloroacetophenone and the product isR-1-(p-chlorophenyl)ethylamine.

Example 33 Production of S-1-(p-chlorophenyl)ethylamine by the ReductiveAmination of p-chloroacetophenone

The method of Example 31 is carried out except that acetophenone isreplaced by p-chloroacetophenone and the product isS-1-(p-chlorophenyl)ethylamine.

Example 34 Production of R-1-(m-bromophenyl)ethylamine by the ReductiveAmination of m-bromoacetophenone

The method of Example 30 is carried out except that acetophenone isreplaced by m-bromoacetophenone and the product isR-1-(m-bromophenyl)ethylamine.

Example 35 Production of S-1-(m-bromophenyl)ethylamine by the ReductiveAmination of m-bromoacetophenone

The method of Example 31 is carried out except that acetophenone isreplaced by m-bromoacetophenone and the product isS-1-(m-bromophenyl)ethylamine.

Example 36 Production of 1-phenylethanol by the Reduction ofAcetophenone

One hundred units of an alcohol dehydrogenase, generated by mutagenesisand screening of the alr1 gene as described in Example 13 above, isincubated at 45EC in 100 milliliters of a solution maintained at pH 6.5containing potassium phosphate (1 millimole), NADPH (0.01 millimole),sodium formate (25 millimoles), and a NADP-utilizing formatedehydrogenase P3 (obtained from Juelich Fine Chemcials, Juelich,Germany; catalog number 25.10; 100 units). Acetophenone (10 millimoles)is added slowly over one hour with stirring, and the reaction is allowedto proceed for an additional 4 hours. The reaction mixture is extractedwith methyl t-butyl ether, and analysis of the reaction products iscarried out by gas chromatography to determine the yield of1-phenylethanol. Chiral analysis is carried out by chiral gaschromatography using a ChiraDex CB column (Advanced SeparationTechnology, Whippany, N.J. USA).

Example 37 Production of R-1-phenylethanol by the Reduction ofAcetophenone

The method of Example 36 is carried out except that the alcoholdehydrogenase is determined to be an R-1-phenylethanol dehydrogenase andthe product is R-1-phenylethanol.

Example 38 Production of S-1-phenylethanol by the Reduction ofAcetophenone

The method of Example 36 is carried out except that the alcoholdehydrogenase is determined to be an 5-1-phenylethanol dehydrogenase andthe product is S-1-phenylethanol,

Example 39 Production of R-1-(p-chlorophenyl)ethanol by the Reduction ofp-chloroacetophenone

The method of Example 36 is carried out except that acetophenone isreplaced by p-chloroacetophenone, the alcohol dehydrogenase isdetermined to be an R-1-(p-chlorophenyl)ethanol dehydrogenase and theproduct is R-1-(p-chlorophenyl)ethanol.

Example 40 Production of S-1-(p-chlorophenyl)ethanol by the Reduction ofp-chloroacetophenone

The method of Example 36 is carried out except that acetophenone isreplaced by p-chloroacetophenone, the alcohol dehydrogenase isdetermined to be an S-1-(p-chlorophenyl)ethanol dehydrogenase and theproduct is S-1-(p-chlorophenyl)ethanol.

Example 41 Production of R-1-(m-bromophenyl)ethanol by the Reduction ofm-bromoacetophenone

The method of Example 36 is carried out except that acetophenone isreplaced by m-bromoacetophenone, the alcohol dehydrogenase is determinedto be an R-1-(m-bromophenyl)ethanol dehydrogenase, and the product isR-1-(m-bromophenyl)ethanol.

Example 42 Production of S-1-(m-bromophenyl)ethanol by the Reduction ofm-bromoacetophenone

The method of Example 36 is carried out except that acetophenone isreplaced by m-bromoacetophenone, the alcohol dehydrogenase is determinedto be an S-1-(m-bromophenyl)ethanol dehydrogenase, and the product isS-1-(m-bromophenyl)ethanol.

The preceding description has been presented with reference to presentlypreferred embodiments of the invention. Workers skilled in the art andtechnology to which this invention pertains will appreciate thatalterations and changes in the described methods and kits may bepracticed without meaningfully departing from the spirit and scope ofthis invention. Accordingly, the foregoing description should not beread as pertaining only to the precise methods and kits described, butrather should be read consistent with and as support to the followingclaims, which are to have their fullest and fair scope.

1. A method for producing an amine from a target ketone, comprising:creating a mutated enzyme that catalyzes reductive amination of thetarget ketone; and providing the mutated enzyme in a reaction mixturecomprising the target ketone under conditions sufficient to permit theformation of the corresponding amine to thereby produce the amine,wherein the ketone is not a 2-ketoacid.
 2. The method of claim 1,wherein the mutated enzyme is an amino acid dehydrogenase.
 3. The methodof claim 2, further comprising providing an existing amino aciddehydrogenase, wherein the mutated enzyme is created by mutating theexisting amino acid dehydrogenase, and further wherein the mutatedenzyme catalyzes reductive amination of the target ketone at a greaterrate than the existing amino acid dehydrogenase.